Estimate the Frequency of Causal Genetic Variants in Fetuses With Congenital Heart Defects: a Chinese Cohort Study

doi:10.21203/rs.3.rs-698990/v1

Purpose: This cohort study was designed to assess the prevalence of chromosomal abnormalities in fetuses with different types of congenital heart defects (CHD).

Methods: In a cohort of 200 fetuses with CHD, we performed prenatal chromosome microarray analysis (CMA) firstly, and then WES analysis was carried out on some fetuses with negative CMA results. Meanwhile, we conducted a systematic literature search on hot spot pathogenic copy number variations (CNVs) related to CHD in the Chinese population.

Results: Chromosomal abnormalities were detected in 49 (24.5%) fetuses after prenatal CMA detection, including 23(11.5%) with aneuploidies and 26 (13.0%) with significant clinical CNVs. The additional diagnostic yield diagnosed by followed WES was 11.5% (6/52). The incidence of total chromosomal abnormality in the non-isolated CHD group (31.8%) was higher than that in the isolated CHD group (20.9%), mainly because the incidence of aneuploidy was significantly increased when CHD combined with extracardial structural abnormalities or soft markers. The chromosome abnormal rate of complex CHD group was higher than that of the simple CHD group, but the difference was not statistically significant (31.8% vs. 23.6%, P = 0.398). The most common CNVs detected in CHD fetuses was the 22q11.2 deletions, followed by deletions of 5p15.33p15.31, deletions of 15q13.2q13.3, deletions of 11q24.2q25, deletions of 17p13.3p13.2, duplications of 17q12.

Conclusion: CMA is the recommended initial examination for cases of CHD in the prenatal setting, whether it is simple heart defects or isolated heart defect. For cases with negative CMA results, the follow-up application of WES will offer a considerable proportion of additional detection of clinical significance.

Internal Medicine

congenital heart defects

chromosomal abnormalities

copy number variations

chromosome microarray analysis

whole exome sequencing

Congenital heart defects (CHD) are the most common congenital birth defects and occur in 5–8 out of 1,000 live births^1,2. The occurrence of congenital heart defect is not only related to genetic factors, but also related to some maternal factors, such as maternal infection with rubella virus, radiation, drug use and environmental pollution.^3,4 With the unprecedented advances in genetic testing techniques, chromosomal abnormalities were thought to be more common causes of CHD than previously thought^5,6.

Known genetic causes include chromosomal abnormalities, chromosome copy number variations and mutations in heart-related genes⁷. Aneuploidies were the earliest identified and the most common genetic causes of CHD⁸. Trisomy 13, trisomy 18, trisomy 21 and Turner syndrome have been widely confirmed to be associated with CHD⁹. Clinically significant copy number variations (CNVs) have been detected in about 10–15% of patients with congenital heart disease¹⁰. Chromosome microarray analysis (CMA) can detect both chromosomal aberrations and copy number variations at the genome-wide level. Currently, CMA has been clinically recommended as the preferred cytogenetic diagnostic test for CHD¹¹. The rapid development of next-generation sequencing technologies, particularly whole exome sequencing (WES), has uncovered mutations that cannot be defined by traditional genomic approaches, opening the door to understanding the genetics of complex diseases such as congenital heart defects^12,13.

At present, there have been some studies on the genetic testing of fetuses with CHD in China. Still, the genotype-phenotype relationship has not yet been fully established, and the hot spot CNVs related to CHD in the Chinese population were still unclear. Here, we present the CMA analysis results of 200 CHD fetuses to evaluate the diagnostic effect of CMA in prenatal diagnosis of CHD and to investigate the possible genetic causes of prenatal CHD cases. Next, we identified new genes associated with CHD through WES and explored the clinical value of WES in prenatal diagnosis. In addition, we performed a systematic literature search to investigate hot spot pathogenic CNVs associated with CHD in the Chinese population. Our study further provides the basis for the standard method of gene detection in CHD fetuses.

Ethics Approval and Consent to Participate

The study protocol was approved by the ethics committee of Changzhou Maternity and Child Health Care Hospital affiliated to Nanjing Medical University. All pregnant women received genetic counseling and signed informed consent before testing.

Subjects

This cohort study was carried out in the department of medical genetics of Changzhou Maternal and Child Health Hospital between January 1st 2015 and August 31th 2020. Pregnant women with fetal with heart defects were referred to our prenatal diagnostic center for further genetic testing counseling. Couples who volunteered for prenatal CMA diagnosis signed written informed consent and underwent invasive prenatal diagnostic procedures. Cases with a positive CMA result received the genetic counseling again. WES was subsequently performed on some cases with negative CMA test results. All fetal specimens were amniotic fluid cells. The average age of pregnant women was 28.26 years (17–46 years), and the average gestational age of fetus was 25.5 weeks (22–30 weeks).

Of the 200 fetuses with congenital heart defects (CHD), 134 with only abnormalities of the heart were classified as the isolated CHD group, and 66 with other extracardiac ultrasound abnormalities, including structural abnormalities and soft markers, were classified as the non-isolated CHD group. Soft markers in this study included increased nuchal fold (≥ 6.0mm), nuchal translucency (≥ 3.0mm), echogenic bowel, mild ventriculomegaly (10-15mm), persistent right umbilical vein, absent nasal bone and single umbilical artery. The detailed anatomical classification of CHD in this study was based on the method described by Botto et al¹⁴. Moreover, cardiac defects were defined as simple CHD (n = 178) if they were anatomically discrete or single or dominant entities¹⁵, and complex CHD (n = 22) if they were characterized by transposition of great arteries, single ventricle or multiple cardiac abnormalities (involving three or more defects).

Chromosomal microarray analysis

CMA analysis was performed using affymetrix Cytoscan 750k array platform. Genomic DNAs were extracted from collected amniotic fluid using the QIAamp DNA Micro Kit. After PCR amplification, DNA was digested and ligated to adapters, and then array hybridization, washing and scanning was subsequently carried. Data was analyzed using the Affymetrix Chromosome Analysis Suite software, based on genome version GRCh37 (hg19). We used the following public databases: PubMed (http://www.ncbi.nlm.nih.gov/pubmed/), ISCA (https://www.iscaconsortium.org/), UCSC (http://genome.ucsc.edu), OMIM (http://www.ncbi.nlm.nih.gov/omim), DECIPHER (http://decipher.sanger.ac.uk/), and DGV (http://www.ncbi.nlm.nih.gov/dbvar/). According to the American College of Medical Genetics (ACMG) guidelines¹⁶, all CNVs reported in this study were classified as VOUS, likely pathogenic (LP), or pathogenic (P). Both likely pathogenic and pathogenic CNVs were considered as clinical significant CNVs in this study.

Whole exome sequencing analysis

Genomic DNA samples were extracted from amniocytes using a Qiagen DNA Blood Midi/Mini kit (Qiagen, Valencia, CA, USA) according to instruction. Agilent SureSelect Human All Exon capture kit (Exome V6) was used for exomes capture and HiSeq2500 sequencer was used for two paired-end massively parallel sequencing. Bcl To Fastq (Illumina) was used to process Raw image files. Low-quality sequenced reads with a quality rating of 20 or less (Q20) were filtered out. SNP analysis, repeated labeling, indel rearrangement, and recalibration of BAM files were performed using SAMtools and Pindel. After using Exome Variant Server databases (http://evs.gs.washington.edu/EVS), dbSNP (http://www.ncbi.nlm.nih.gov/snp), 1000 Genomes Project (1000 GP) (http://browser.1000genomes.org) and Genome Aggregation Databases (gnomAD) (http://gnomad.broadinstitue.org/), minor allele frequencies (MAFs) of all known variants were reported. The perniciousness and pathogenicity of mutations were determined by the following databases: OMIM, ClinVar, Human Gene Mutation Database and SwissVar. In order to analyze the biological effects and evaluate the pathogenicity of all the detected variants, the following prediction software was used: SIFT, Mutation Taster, PolyPhen-2, PROVEAN, CADD, Human Splicing Finder, And MaxEntScan. Sanger sequencing was used to confirm the selected candidate pathogenic variants. According to the American College of Medical Genetics and Genomics (ACMG) guidelines¹⁷, all the selected variants were interpreted as pathogenic, likely pathogenic, VOUS, or benign.

Systematic literature search

To investigate hot spot pathogenic CNVs associated with CHD in the Chinese population, we performed a systematic literature search according to the PRISMA guidelines¹⁸, when applicable. We searched PubMed (https://pubmed.ncbi.nlm.nih.gov/) for all published cohort studies that reported CMA results of fetal congenital heart defects from February 2015 to February 2020 using the following search string: ((“CHD” OR “congenital heart defect” OR “congenital heart disease”) AND (“CMA” OR “CNVs” OR “genetic diagnoses” OR “chromosomal microarray analysis” OR “copy number variants” OR “Genetic Testing”)) NOT review [pt] AND English [la].

Studies were included if they met the following criteria: (1) More than 99 samples were included in the study. (2) The subjects of the study were Chinese. (3) Detailed chromosomal loci of pathogenic and likely pathogenic CNVs were clearly described.

Statistical analyses

SPSS software (IBMSPSS statistics version 14.0) was used for statistical analysis. The chromosomal abnormalities or aneuploidies or CNVs detection rates in isolated CHD, non-isolated, simple CHD and complex CHD were pairwise compared using fisher’s exact test or chi square test. In all tests, P < 0.05 was defined as statistically significant.

Etiology of CHD

Overall, 200 fetuses were included in the CMA test. Of which, 134 were isolated CHD and 66 were non-isolated CHD, including structural anomalies (n = 28), soft markers (n = 22), structural anomalies & soft markers (n = 16). Moreover, 178 of the 200 fetuses were simple CHD and 22 were complex CHD. In addition, according to the anatomical classification proposed by Botto et al., CHD were divided into eight main groups. Among them, the three most common heart abnormalities were septal defects (60/200, 30.0%), conotruncal defects (49/200, 24.5%) and left ventricular outflow tract defects (29/200, 14.5%).

After prenatal CMA testing, chromosomal abnormalities were detected in 49 fetuses, the prevalence was 24.5% (49/200). Among them, 23 cases (11.5%) with aneuploidies, including 8 case for trisomy 21 (T21), 9 for trisomy 18 (T18) and 6 for trisomy 13 (T13). Additionally, clinical significant CNVs were detected in 26 (13%) cases, including 20 (10.0%) as pathogenic CNVs and 6 (3.0%) as likely pathogenic CNVs. Besides, VOUS CNVs were detected in 8 (4%) cases, the other 143 fetuses reported as negative CMA results. Finally, 52 cases were recalled again and received WES test further after genetic counseling, of which 6 (11.5%) were found to have pathogenic or likely pathogenic sequence variant. The process and brief results of the study were shown in Fig. 1.

Subgroup analysis of different types of CHD

Next, we discussed the relationship between chromosomal abnormalities and CHD types. Firstly, compared with isolated CHD group, the chromosome abnormality rate and aneuploidy rate of non-isolated CHD group was higher, but the clinical significant CNVs rate was lower. As shown in Table 1, the chromosomal abnormalities rate of the non-isolated CHD group was 31.8% (21/66), and that of the isolated CHD group was 20.9% (28/134). It's worth noting that the increase of chromosome abnormality rate in non-isolated CHD group was mainly because the incidence of aneuploidy was significantly increased when CHD combined with extracardial structural abnormalities or soft markers. Secondly, the chromosome abnormality rate of complex CHD group was higher than simple CHD group (31.8% vs. 23.6%), including aneuploidies (13.6% vs. 11.2%) and clinical significant CNVs (18.2% vs. 12.4%). Thirdly, the incidence of fetal chromosomal abnormality was most higher in fetuses with atrioventricular septal defects (AVSD) (54.5%). The chromosome abnormality rate of different subgroups of CHD are shown in Table 2.

Subsequently, we discussed the association between the incidence of chromosomal abnormality and extracardiac structural abnormalities. All fetuses with extracardiac structural abnormalities were included, regardless of whether they had soft markers. Therefore, a total of 44 fetuses with extracardiac structural abnormalities were included, including 36 fetuses with single extracardiac structural abnormality and 8 fetuses with multiple extracardiac structural abnormalities. Among all 44 cases, the incidence of chromosomal abnormality was 34.1% (15/44), aneuploidies were found in 18.2% (8/44), and clinical significant CNVs were found in 15.9% (7/44). Additionally, we found that there was no statistical difference in the incidence of chromosomal abnormality in fetuses with single extracardiac structural abnormality and multiple extracardiac structural anomalies (33.3% vs. 37.5%, p > 0.05). Moreover, in these cases of CHD with extracardiac structural abnormalities, the cases with central nervous system abnormalities have a higher probability of chromosomal abnormalities. Table 3 summarized the detection of chromosomal abnormalities in cases of CHD with different types of extracardiac structural abnormalities.

Finally, we also analyzed the association between the incidence of chromosomal abnormality and soft markers. In 38 cases of CHD with soft markers, 34 cases had single soft marker, 4 cases had multiple soft markers. CHD combined with single umbilical artery and nasal bone absent/shortened were the most common. The detection rate of chromosomal abnormality was 45.7% (13/38) in CHD fetuses with soft markers. The incidence of aneuploidy in the CHD fetus with soft markers was (26.3%, 10/38) higher than that of clinical significant CNVs (7.9%, 3/38). Our data suggested that CHD fetuses with soft markers had higher chance of detecting aneuploidies, especially with absent or shortened nasal bone (71.4%, 5/7). Moreover, combining multiple soft markers did not increase the chromosomal abnormalities (35.3% vs. 25%, p > 0.05). The chromosomal abnormalities of CHD with soft markers are shown in Table 4. In addition, no significant difference was observed in the chromosomal abnormalities rates between CHD with extracardiac structural anomalies and CHD with soft markers groups (34.1% vs. 45.7%, p = 0.991). Notably, the chromosome abnormality rate of CHD combined only soft makers group, CHD combined only structural anomalies group and CHD combined both soft makers group and structural anomalies group were 27.3%, 28.6% and 43.8%, respectively. This suggests that the incidence of chromosomal abnormality is greatly increased in CHD fetuses with both soft markers and additional structural anomalies (Table 1).

WES analysis

After informed consent, 52 CHD fetuses with negative CMA test were further analyzed by WES, including 44 cases of isolated CHD and 8 cases of non-isolated CHD. As show in Table 5, a total of 18 cases with 22 sequence variants which fulfilled the filtering criteria were detected. 3 (5.8%) cases with pathogenic sequence variants and 3 (5.8%) cases with likely pathogenic sequence variants. The additional diagnostic yield of clinical significant sequence variants by WES testing for fetuses with CHD was 11.5% (6/52). Frequently encountered gene included NOTCH1, GLI3, DNAH, SCN5A.

Hotspot significant CNVs related to CHD in Chinese population

In order to explore the characteristics of clinical significant CNVs associated with CHD in the Chinese population, we conducted a systematic literature retrieval analysis in addition to analyzing our results. Five papers met our criterion were selected for a detailed full-text review.

We summarized and analyzed CMA data from 200 cases in our study and 1385 cases reported in 5 other literature reports (Table 6). A total of 161 pathogenic or likely pathogenic CNVs were found in 9.0% of cases (143/1585). All chromosomes, except 14, 19 and Y, had clinical significant CNVs, and the clinical significant CNVs on chromosomes 22, 16 and 15 were the most common. The deletions of 22q11.2 was the most common clinical significant CNVs, accounting for 27.3% (44/161). In fetuses with 22q11.2 deletion, the most common heart defects were Tetralogy of Fallot (52.3%, 23/44), ventricular septal defect (27.3%, 12/44), and interrupted aortic arch (18.2%, 8/44). The other 5 most commonly recurrent CNVs loci related to CHD were deletions of 5p15.33p15.31(Cri du chat syndrome), deletions of 15q13.2q13.3 (Angelman/Prader-Willi syndrome), deletions of 11q24.2q25 (Jacobsen syndrome), deletions of 17p13.3p13.2 (Miller-Diekers syndrome), duplications of 17q12. In addition, Fig. 2 showed all of the clinical significant CNVs from our study and the literatures. All CNVs found by CMA in this cohort were listed in Table 7.

In this study, we used a combination of chromosome microarray analysis (CMA) and whole exome sequencing (WES) analysis to assess the prevalence of genetic diagnosis in fetuses with congenital heart defects. Chromosomal abnormalities were found by CMA in 24.5% of CHD fetuses. In addition, the additional diagnostic yield of clinical sequence variants by WES testing for fetuses with CHD was 11.5% (6/52). Many studies have reported the detection rate of CMA in fetuses with congenital heart defect, but the detection rate of CMA varies due to the different array types used¹⁹. It is reported that the positive rate of CMA test in prenatal evaluation is 6.6% − 38.7%^20–22. Overall, the yield of the CMA test in our study was consistent with previously published data. However, the detection rate of our pathogenic sequence variants is higher than that reported in the literature^23–25. In conclusion, we suggest CMA as a first-tier test in fetuses with CHD, and that WES can be offered sequentially if CMA results are negative.

It is widely believed that if there is an additional deformity in the fetus with congenital heart disease, it will significantly increase the possibility of potential genetic causes^26,27. In the present study, the chromosome abnormalities detection rates for isolated CHD, non-isolated CHD, CHD plus structural anomalies, CHD plus soft markers and CHD plus structural anomalies & soft markers were 20.9%, 31.8%, 28.6%, 27.3% and 43.8%, respectively. From the detection rate of isolated and non-isolated CHD cases, the detection rate of non-isolated CHD fetus was higher than that of isolated CHD fetus (31.8% vs. 20.9%), but the difference was not statistically significant (P > 0.05). This result was consistent with what has reported by Liao et al¹⁵. However, we did observe that the incidence of chromosomal abnormality was significantly higher when CHD was combined with structural abnormalities and soft markers. Also, in our study, the detection rate in complex CHD group (31.8%) was higher than but not statistically different from simple CHD group (23.6%). Rivka's study also showed that there was no significant difference in the incidence of chromosomal abnormality between simple and complex CHD²⁸. This suggests that the complexity of heart defects is not related to the frequency of chromosome abnormalities. Therefore, we suggest that CMA is the recommended initial examination for cases of CHD in the prenatal setting, including simple heart disease and isolated heart disease.

Furthermore, our results showed that compared with other types of structural abnormalities, the incidence of chromosomal abnormality in CHD with central nervous system abnormalities was significantly increased, this phenomenon was also reported by Richards.³¹ Previous studies have reported that soft markers indicate an increased risk of fetal chromosomal aneuploidy. Indeed, in our study, the detection rate of aneuploidies in CHD with soft markers group is as high as 22.7%, which is significant higher than that in isolated CHD group and CHD with extracardiac structural anomalies group. Among all CHD groups, AVSD (54.5%) had the highest detection rate of chromosomal abnormalities, followed by complex CHD (31.8%), and conotruncal defect (24.5%). Similar results have been obtained in previously published data. Wang et al. reported that among 602 cases of CHD detected by CMA, the most common chromosomal abnormalities were observed in fetuses with AVSD (73.7%)²³. Although the subgroup size of our cohort was small, we got very similar results with Wang's study.

We not only analyzed the data from our center for this cohort study, but also systematically reviewed the literatures on clinically significant CNVs reported by CMA in Chinese CHD fetuses. In our study, the overall detection rate of chromosomal abnormality was 24.5% (49/200), and the total detection rate for clinical significant CNVs was 13.0% (26/200). Moreover, the detection rate of total chromosome abnormalities in 1385 CHD fetuses summarized in the literatures was 25.1% (348/1385), and the detection rate of clinical significant CNVs was 8.4% (117/1385). Overall, the incidence of pathogenic chromosomal abnormality and clinical significant CNVs in CHD fetuses in our study were similar to what has been reported in the literatures. Studies have shown that the deletion of 22q11.2 is associated with cardiac abnormalities²⁹. Indeed, both in our study and reported in the literatures, the most common CNVs detected in CHD fetuses was in the 22q11.2 region. Additionally, 22q11.2 deletion was reported to be associated with tetralogy of Fallot by Mercer-Rosa et al ³⁰. In our study, 52.3% of fetuses with 22q11.2 deletions had Tetralogy of Fallot. In the combined data from the literature, 47.7% of fetuses with 22q11.2 deletions had Tetralogy of Fallot.

This study not only complements the previous data that CMA plays an important role in prenatal detection of CHD, but also evaluated the yield of CMA in regard to specific clinical characteristic, and even discusses the clinical value of WES in prenatal diagnosis of CHD.

CMA is the recommended initial examination for cases of CHD in the prenatal setting, including simple heart disease and isolated heart disease. The follow-up application of WES will offer a considerable proportion of additional detection of clinical significance. 22q11.2 was the most common pathogenic region of Chinese population.

Ethics approval and consent to participate

The study design and protocol were reviewed and approved by the Ethics Committee of the Changzhou Maternity and Child Health Care Hospital, Nanjing Medical University.

Consent for publication

The parents of the patients participating in this research provided written informed consent.

Availability of data and materials

The questionnaire and datasets used are available from the corresponding author on request.

Authors' contributions

FYL and JW collected the clinical data, analyzed the data, and wrote the manuscript. JBL and PX carried out clinical consultations and physical examination. BY and BZ conceived the study, participated in its design and coordination.

Competing interests

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Funding

This study was funded by the Changzhou Key Laboratory of High-tech Research (grant number CM20193009), Changzhou Social Development Science and Technology Support Project (grant number CE20205035), Jiangsu Maternal and Children Healthcare Key Discipline (FXK201754)，Science and Technology Development Fund of Nanjing Medical University (NMUB2019320).

Acknowledgments

We are thankful to the patient and the family for their participation in the study.

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Table 1

Distribution of genetic variants in different group
CHD group	n	Chromosomal abnormalities
CHD group	n	Aneuploidies	Clinical significant CNVs	Total
Isolated CHD	134	10 (7.5%)	18 (13.4%)	28 (20.9%)
Non-isolated CHD	66	13 (19.7%)*	8 (12.1%)	21 (31.8%)
CHD plus structural anomalies	28	3(10.7%)	5(17.9%)	8(28.6%)
CHD plus soft marker	22	5(22.7%)*	1(4.5%)	6(27.3%)
CHD plus structural anomalies & soft marker	16	5(31.3%)*	2(12.5%)	7(43.8%)
Simple CHD	178	20(11.2%)	22(12.4%)	42(23.6%)
Complex CHD	22	3(13.6%)	4(18.2%)	7(31.8%)
Note: *: Compared with isolated CHD, p < 0.05.
CHD, congenital heart disease; CNV, copy number variation;

Table 2

Types of congenital heart disease and frequencies for fetuses with chromosomal abnormalities.
CHD classfication	No.tested	Aneuploidies	significant CNVs	Total	DR(%)
Septal defects	60	7	5	12	20.0
VSD	55	6	4	10	18.2
VSD + ASD	5	1	1	2	40.0
Conotruncal defects	49	4	8	12	24.5
Truncus arteriosus	4	0	0	0	0
IAA	8	0	2	2	25.0
d-TGA	8	0	1	1	12.5
DORV	11	1	2	2	18.2
TOF	18	3	4	7	38.9
LVOTO	29	4	3	7	24.1
HLHS	11	1		1	9.1
Coarctation of aorta	10	3	2	5	50.0
Aortic stenosis	8	0	1	1	12.5
RVOTO	19	1	3	4	21.1
Pulmonary stenosis	12	1	2	3	25.0
Pulmonary atresia	2	0	1	1	50.0
Tricuspid atresia	5	0	0	0	0
AVSD	11	4	2	6	54.5
Complex CHD	22	3	4	7	31.8
Multiple, complex heart anomaly	19	3	4	7	36.8
Single ventricle	2	0	0	0	0
L-TGA	1	0	0	0	0
Heterotaxy	2	0	0	0	0
Other CHD	8	0	0	1	12.5
Total	200	23	26	49	24.5
CHD, congenital heart disease; NCA, numerical chromosomal abnormality; DR, detection rate; CNV, copy number variation; pCNV, pathogenic copy number variation;VSD, ventricular septal defect; ASD, atrial septal defect; AVSD, atrioventricular septal defect; DORV, double outlet right ventricle; d-TGA, d-transposition of great arteries; IAA, interrupted aortic arch; LVOTO, left ventricular outflow tract obstruction; RVOTO, right ventricular outflow tract obstruction.

Table 3

Detection rates of chromosomal abnormalities in fetuses with CHD plus additional structural anomalies
CHD with additional structural anomalies	n	Chromosomal abnormalities
CHD with additional structural anomalies	n	Total	Aneuploidy	pCNV
CHD with single additional structural anomaly	36	12	6	6
Central nervous system	3	2	1	1
Gastrointestinal system	2	1	1	0
Urinary tract system	4	1	0	1
Respiratory system	2	0	0	0
Skeletal system	10	4	3	1
Face	8	3	1	2
Cystic hygroma	6	1	0	1
Abdominal wall	1	0	0	0
CHD with multiple additional structural anomalies	8	3	2	1

Table 4

Detection rates of chromosomal abnormalities in fetuses with CHD plus soft markers
CHD with nonstructuralanomalies	n	Chromosomal abnormalities
CHD with nonstructuralanomalies	n	Total	Aneuploidy	pCNV
CHD with single soft marker	34	12	9	3
Single umbilical artery	11	3	1	2
Absent or shortened nasal bone	7	5	5	0
Mild ventriculomegaly	4	0	0	0
Short long bones	1	0	0	0
Echogenic bowel	1	1	0	1
Persistent right umbilical vein	3	0	0	0
Thickened nuchal fold	0	0	0	0
Enlarged cisterna magna	1	0	0	0
Increased nuchal translucency	0	0	0	0
Choroid plexus cysts	6	3	3	0
Pyelectasis	0	0	0	0
CHD with multiple soft markers	4	1	1	0

Table 5

Detection of pathogenic and likely pathogenic variants in fetuses with CHD using WES
Case	Ultrasound findings	Additional anomalies	Gene	Nucleotide change	Amino acid change	Zygosity	Clinical classify	Inherited mode	Disease
#133	RAA	—	GLI3	c.2308delG	p.A770fs	Het	LP	AD	Pallister-Hall syndrome
#79	AVSD	—	SCN5A	c.4357C > T	p.Q1453X	Het	LP	AD	Brugada syndrome 1
#25	VSD	Urinary tract system,Skeletal system	NIPBL	c.5220delA	p.T1740fs	Het	P	AD	Cornelia de Lange syndrome 1
#159	IAA, AVSD	—	FOXF1	c.853_854delAT	p.Ile285fs	Het	P	AD	Persistent fetal circulation syndrome
#170	VSD	—	SCN5A	c.362G > A	p.Arg121Gln	Het	P	AD	Brugada syndrome 1
#166	PA	—	GATA6	c.551G > A	p.Ser184Asn	Het	LP	AD	Atrial septal defect 9
#62	VSD,d-TGA,TA	—	MYH7	c.4076G > A	p.R1359H	Het	VUS	AD	—
#19	VSD,CA	—	CITED2	c.589A > G	p.S197G	Het	VUS	AD	—
#132	VSD	Mild ventriculomegaly	NOTCH1	c.7171C > T	p.Q2391X	Het	VUS	AD	—
#161	AS	—	NOTCH1	c.5339_5346dupAGAAGAAG	p.Glu1785fs	Het	VUS	AD	—
#168	VSD	—	COL9A3	c.622G > A	p.Gly208Ser	Het	VUS	AD	—
#171	TOF	—	PRNP	c.622C > T	p.Arg208Cys	Het	VUS	AD	—
#192	VSD	—	HDAC	c.1 + 1G > A		Het	VUS	AD	—
#194	heterotaxy	Cystic hygroma	DNAH9	c.1997G > T	p.Trp666Leu	Het	VUS	AD	—
#200	heterotaxy	—	DNAH9	c.14_34delAGGAGCGGGCCGCGC	p.Glu5_Ala11del	Het	VUS	AD	—
#98	TOF	—	CCD22	c.664C > T	p.R222W	Het	VUS	AD	—
#106	VSD, CA	—	PRKD1	c.2569G > C	p.E857Q	Het	VUS	AD	—
		Skeletal system,	MYH6	c.4036C > T	p.R1346W	Het	VUS	AD	—
		—	COL5A1	c.2768C > T	p.P923L	Het	VUS	AD	—
#51	HLHS	—	GLI3	c.2587C > T	p.R863C	Het	VUS	AD	—
#101	VSD	Thickened nuchal fold	SOS2	c.3812C > T	p.P1271L	Het	VUS	AD	—
Het, heterozygous; AD, autosomal dominant; AR, autosomal recessive; LP, likely pathogenic; P, pathogenic; VUS, variants of uncertain significance. VSD, ventricular septal defect; TGA, transposition of the great arteries; CA, coarctation of aorta; RAA, right aortic arch; AVSD, atrioventricular septal defect; TOF, tetralogy of fallot; HLHS, hypoplastic left heart syndrome; IAA, interruption of aortic arch; AS, aortic stenosis; PA, pulmomary stenosis; transposition of the great arteries; CA, coarctation of aorta; RAA, right aortic arch; AVSD, atrioventricular septal defect; TOF, tetralogy of fallot; HLHS, hypoplastic left heart syndrome; IAA, interruption of aortic arch; AS, aortic stenosis; PA, pulmomary stenosis.

Table 6

The comparison of studies in prevalence of genetic variants identified in CHD fetuses by CMA.
Study	Platform	Number	Total chromosomal abnormalities	Aneuploidies	Partial Aneuploidies	pCNVs	lpCNVs	Vous
Song et al. 2018 [18]	Affymetrix Cytoscan 750k array	207	35 (16.9%)	17 (8.2%)	-	13 (6.3%)	5 (2.4%)	14 (6.8%)
Luo et al. 2018 [19]	Illumina HumanCytoSNP-12 v2.1 BeadChip	362	140 (38.7%)	111 (30.7%)	10 (2.8%)	17 (4.7%)	2 (0.6%)	-
Zhu et al. 2016 [20]	AffymetrixCytoScanplatform	115	21 (18.3%)	6 (5.2%)	2 (1.7%)	11 (9.6%)	2 (1.7%)	-
Wang et al. 2017 [21]	Illumina HumanCytoSNP-12 v2.1 BeadChip	602	133 (22.1%)	65 (10.8%)	20 (3.3%)	40 (6.7%)	8 (1.3%)	36 (6.0%)
Liao et al. 2014 [22]	Affymetrix CytoScan HD arrays	99	19 (19.2%)	excluded by CK	excluded by CK	19 (19.2%)	-	13 (13.1%)
Our study	Affymetrix Cytoscan 750k array	200	49 (24.5%)	23 (11.5%)	-	20 (10.0%)	6 (3.0%)	8 (4%)
CK: conventional karyotype; pCNVs: pathogenic copy number variation; lpCNVs: likely pathogenic copy number variation.

Table 7

Pathogenic or likely pathogenic CNVs found in the cohort.
CASE	CHD Phenotype	Soft markers	Structural anomalies	CNV	Microarray Nomenclature	Size (Mbp)	Pathogenicity	Some of Relevant Genes	syndrome
#6	VSD,	—	facial dysmorphisms	del	arr[hg19] 3q29(194654896–197363564)x1	2.71	P	TFRC	Chromosome 3q29 microdeletion syndrome
#20	TOF	—	respiratory system	del	arr[hg19] 22q11.1q11.21(17900000–20600000)x1	2.7	P	ATP6V1E1, PEX26,TUBA8	22q11 deletion syndrome
#23	PVS	—	—	del	arr[hg19] 2p25.3(50-450000)x1	0.45	P	—	—
#24	Complex CHD	—	—	dup	arr[hg19] 15q24.3q25.2(78250000–85000000)x1	6.75	P	ARNT2, RPS17,AP3B2	Chromosome 15q25 deletion syndrome
#35	Complex CHD	—	—	del	arr[hg19] 5p15.33p14.2(20000-23980000)x3	23.96	P	SDHA, NDUFS6	mitochondrial complex II deficiency, Leigh syndrome(LS)
#35	Complex CHD	—	—	dup	arr[hg19] 21(q22.12-q22.3)(37780000–48100000)x3	10.32	P	—	Down syndome
#39	PAA	—	—	del	arr[hg19] 7q11.23(72260000–76000000)x3	3.74	P	POR	7q11.23 duplication syndrome, WILLIAMS-BEUREN region duplication syndrome
#41	d-TGA	—	—	dup	arr[hg19] 1p36.33p36.32(820000–3320000)x3	2.5	LP	AGRN,B3GALT6, TMEM240	congenital myasthenic syndrome-8 (CMS8), Ehlers-Danlos syndrome progeroid type 2
#56	DORV, PAA	—	—	del	arr[hg19] 5p15.33p15.1(20000-16500000)x1	16.48	P	SDHA	5p15 terminal (Cri du chat syndrome) region duplication,mitochondrial complex II deficiency[OMIM:252011]
#56	DORV,PAA	—	—	dup	arr[hg19] 17q24.2q25.3(65320000–81160000))x3	15.84	P	PSMD12	46 XX sex reversal 2, STANKIEWICZ-ISIDOR syndrome(STISS)
#59	AVSD	—	—	del	arr[hg19] 20p12.3(6300001–8580000)x3	2.28	LP	BMP2	BRACHYDACTYLY, TYPE A2(BDA2), Early infantile epileptic encephalopathy-12 (EIEE12)
#78	VSD + ASD	Single umbilical artery	facial dysmorphisms	del	arr[hg19] 7p22.3p22.1(40000-6740000)x1	6.7	LP	BRAT1	lethal neonatal rigidity and multifocal seizure syndrome
#78	VSD + ASD	Single umbilical artery	facial dysmorphisms	dup	arr[hg19] 12p13.33p13.31(160000–8320000)x3	8.16	LP	CACNA1C, CCND2, CHD4	Timothy syndrome(TS), SIFRIM-HITZ-WEISS syndrome (SIHIWES)
#82	TOF	—	—	dup	arr[hg19] 8p23.1(6920000–12580000)x3	5.66	P	—	8p23.1 duplication syndrome
#84	TOF	—	—	del	arr[hg19] 22q11.21(18880000–21440000)x1	2.56	P	PRODH	DIGEORGE syndrome; DGS (proximal, A-B)syndrome1,
#85	VSD, IAA	—	—	del	arr[hg19] 22q11.21q11.23(18564800–24300000)x1	3.2	P	PEX26, TUBA8,PRODH	22q11 deletion syndrome
#100	TOF	—	—	del	arr[hg19] 22q11.21(18,970,561 − 21,800,471)x1	2.8	P	CLTCL1, HIRA,TBX1	DiGeorgesyndrome(DGS), velocardio facial syndrome༈VCFS༉
#110	Other CHD	—	central nervous	del	arr[hg19] 7q33q36.3(137,754,586 − 159,119,707)x1	21.4	P	BRAF, CNTNAP2, DPP6	cardiofaciocutaneous syndrome-1, Noonan syndrome 7,
#110	Other CHD	—	central nervous	dup	arr[hg19] 20q13.2q13.33(51,222,942 − 62,913,645)x3	11.7	P	CYP24A1, PCK1,OSBPL2, RTEL1	HYPERCALCEMIA INFANTILE-1(HCINF1), phosphoenolpyruvate carboxykinase deficiency
#111	VSD	—	—	del	arr[hg19] 16p11.2(29,428,531 − 30,190,029)x1	0.74	LP	PRRT2, KCTD13, TBX6, MAPK3	16p11.2deletion syndrome
#114	AS	—	Skeletal system	del	arr[hg19] 22q11.21(18,631,364 − 21,800,471)x1	3.2	P	CLTCL1,HIRA,TBX1	DiGeorgesyndrome(DGS), velo- cardio- facial syndrome(VCFS)
#116	CA, VSD, PLSVC	—	Cystic hygroma, Skeletal system	del	arr[hg19] 4p16.3p15.31(68,345 − 18,451,423)x1	18.4	P	LONP1, NFIX, SMARCA4	BWS/SRS(Beckwith–Wiedemann syndrome/Silver-Russell syndrome;CODAS syndrome
#116	CA, VSD, PLSVC	—	Cystic hygroma, Skeletal system	dup	arr[hg19] 11p15.5p15.1(230,680 − 20,167,667)x3	19.9	P	APC2,TICAM1, LONP1	Pigmented nodular adrenocortical diseaseprimarysyndrome; CODAS syndrome
#117	Other CHD	Echogenic bowel	Skeletal system	del	arr[hg19] 15q11.2(22,770,421 − 23,277,436)x1	0.50	LP	TUBGCP5, CYFIP1, NIPA2, NIPA1	15q11.2 recurrent region (BP1-BP2) (includes NIPA1) deletionsyndrome1
#120	Other CHD	Choroid plexus cysts	facial dysmorphisms	dup	arr[hg19] Xp22.31(6,455,151-8,135,568)x3	1.68	P	PUDP, STS, VCX, PNPLA4	Steroid sulphatase deficiency (STS)
#121	PAA,ASD	—	urinary tract system, facial dysmorphisms	del	arr[hg19] 4q31.3(151,335,416 − 151,834,016)x1	0.5	LP	LRBA, MAB21L2	—
#135	CA	—	—	del	arr[hg19] 21q22.2q22.3(39,985,071 − 48,093,361)x1	8.1	P	KPTN	autosomal recessive mental retardation-41 (MRT41)
#136	VSD	—	—	dup	arr[hg19] 9q34.11q34.3(133,343,703 − 141,018,648)x3	7.7	P	—	—
#138	AVSD	—	—	del	arr[hg19] 16q24.1q24.2(85,520,919 − 87,149,214)x1	1.6	P	FOXF1, FOXC2, FOXL1	—
#157	VSD, IAA	—	—	del	arr[hg19] 22q11.21(18,636,749 − 21,800,471)x1	3.16	P	CLTCL1, HIRA,TBX1	DiGeorgesyndrome(DGS), velo- cardio- facial syndrome༈VCFS༉
#179	VSD	—	—	del	arr[hg19] 15q11.2(22,770,421 − 23,625,785)x1	0.83	LP	TUBGCP5, CYFIP1, NIPA2, NIPA1	15q11.2 recurrent region (BP1-BP2)deletionsyndrome
#179	VSD	—	—	del	arr[hg19] 16p13.11(14,910,158 − 16,520,463)x1	1.6	P	NOMO1, NPIPA1, PDXDC1, NTAN1 ), RRN3, KIAA0430, NDE1, MYH11, ABCC1, ABCC6, NOMO3	16p13.11 recurrent microdeletion syndrome
CNVs found by CMA in the cohort, with the number of genes present in the region, listing the most relevant genes and phenotypes for each individual. Dup, Duplication; Del, Deletion; CA, congenital anomalies; LP, likely pathogenic; P, pathogenic; VOUS, variants of uncertain significance. VSD, ventricular septal defect; TGA, transposition of the great arteries; CA, coarctation of aorta; RAA, right aortic arch; ASD, atrial septum defect; AS, aortic stenosis; AVSD, atrioventricular septal defect; TOF, tetralogy of fallot; HLHS, hypoplastic left heart syndrome; IAA, interruption of aortic arch; PLSVC: persistent left superior vena cava;PAA: pulmonary artery atresia; PVS: pulmonary valve stenosis; DOLV: double outlet right ventricle

Estimate the Frequency of Causal Genetic Variants in Fetuses With Congenital Heart Defects: a Chinese Cohort Study

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Abstract

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Introduction

Methods

Results

Discussion

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